Cholea is a pandemic disease caused by V. cholerae in which cholera toxin (CT) cause life-threatening diarrhea. Enterotoxigenic E. coli and other enteroxic enteric bacteria cause diarrheal disease closely related to cholera. Highly effective vaccines against these diseases are not yet available. Our long range goals are to understand the structure and function of cholera toxin (CT) at the molecular level and to exploit the remarkable properties of holotoxin diseases. Specific Aim 1 will use biochemical, genetic, cell biological, and structural methods to characterize features of CT-A and CT-B that determine the biological actives of CT. These studies will analyze interactions between CT-A and CT-B that are essential for assembly of CT, determine structure of conformation-dependent epitopes of CT-B that elicit neutralizing antibodies, investigate the activation pathway for CT-A and determine the structure of active CT-A1, explore the role of specific gangliosides in intracellular trafficking of CT and related enterotoxins, and develop assays for translocation of CT-A across intracellular membranes. Specific Aim 2 will evaluate holotxin-like chimeras, in which microbial protective antigens replace the A1 domain of CT, as model oral factors of Vibrio Cholerae, will be tested for ability to induce protective anti-toxic and anti-bacterial immunity against cholera; and chimeras that incorporate Streptococcus pneumoniae PspA, which express conserved protective epitopes, will be tested for their ability to induce cross-protective immunity against pneumococci of multiple capsular serotypes.